Chronic wound remains as a constant challenge for patients with diabetes or treated with immunosuppressive drugs, chemo- and radio-therapies. Mechanisms underlying poor wound healing are still unclear (see, Fang, et al., Nutrition. 2002, 18, pp. 872-879). Wound healing is generally characterized by haemostasis, inflammation, migration and proliferation of fibroblasts, endothelial and epithelial cells, deposition of connective tissue such as collagen, followed by angiogenesis and re-epithelialization. A complex cascade of regulated steps with complications of persistent inflammation, fibroblast dysfunction and impaired angiogenesis often leads to morbidity and mortality.
An increased level of oxygen free radicals and other reactive oxygen species (ROS) like H2O2 as by-products along with pro-inflammatory mediators at wound site, play a crucial role in impaired wound healing. Hence, antioxidants that help in maintaining a balance by metabolizing/reducing the ROS levels at wound site could be an effective strategy to ameliorate cellular dysfunction.
Apart from pharmacological approaches such as curcumin, a known antioxidant and anti-inflammatory agent with poor bioavailability issues (see, Anand, P et al., Mol. Pharmaceut., 2007, 4, pp. 807-818), conventional surgical treatments like skin autograft and allograft, artificial skin embedded with epidermal/fibroblastic cells has their own disadvantages such as limited tissue availability, high cost, immune-rejection and prolonged time of healing. To overcome these limitations, bone marrow derived stem cells can be potential candidates for cell transplantation therapies (see, Schafer, M et al., Pharmacol. Res. 2008, 58, pp. 165-171). Transplantation of stem cells isolated from bone marrow has been used for tissue regeneration, albeit with a limitation of low bio-availability upon systemic administration (see, Chen, F. M et al., Biomaterials 2011, 32, pp. 189-209). Nevertheless, this strategy, has also met with limited success owing to enhanced ROS (Reactive oxygen species) levels at injury site that impairs the function of stem cells transplanted at chronic wounds (see, Case, J. et al Antioxid. Redox Signal. 2008, 10, pp. 1895-1907). Thus, a delivery mechanism of stem cells on-site coupled with capabilities of ROS levels diminution could be an improved and novel therapeutic strategy for chronic wounds.
To protect transplanted cells, use of biodegradable scaffolds/bio-polymers as carriers of cells to the site of tissue injury could be an efficient therapeutic approach. Synthetic biodegradable polymers have proven to be highly potent in many biomedical applications. In tissue engineering, these polymers can be used as temporary scaffolds and as surgical devices or implants for drug and gene delivery (see, Crisanti, C. M. et al., J. Surg. Res. 2008, 146, pp. 3-10). Polymer material should be processed into thin films with a controlled thickness, biocompatible, allow attachment/proliferation of anchorage-dependent cells, and bio-degradable over a desired period of time after implantation (see, Seal B. L et al., Mater. Sci. Eng: Reports 2001, 34, pp. 147-230; WO1994025079A1, U.S. Pat. No. 5,723,508, U.S. Pat. No. 5,686,091, U.S. Pat. No. 5,514,378, U.S. Pat. No. 5,198,507, U.S. Pat. No. 5,099,060, U.S. Pat. No. 4,826,945, U.S. Pat. No. 6,103,255A).
Currently, aliphatic polyesters prepared from lactic and glycolic acids are the most versatile and widely used synthetic biodegradable polymers as suture material, scaffolds for soft and hard tissue repair as well as drug carriers. (See, U.S. Pat. No. 6,626,950, U.S. Pat. No. 6,485,521, U.S. Pat. No. 6,696,575, U.S. Pat. No. 6,696,575, U.S. Pat. No. 6,743,232, U.S. Pat. No. 6,852,330, U.S. Pat. No. 7,101,857, U.S. Pat. No. 7,338,517, Sabir, M. I et al., J. Mater. Sci. 2009, 44, pp. 5713-5724.; Chi-KuangFeng, Y. L et al., J. Med. Biol. Eng. 2001, 21, pp. 233-242; Liao, S. S et al., J Bioact. Compat. Polym. 2004, 19, pp. 117-130). However, contradictory reports exist regarding the biocompatible properties of these PLA-PGA-based biopolymers, (see, Makadia, H. K et al., Polymers 2011, 3, pp. 1377-1397; Dhandayuthapani, B et al., Int. J Polym. Sci. 2011, 9) such as, release of acidic by-products, poor processability, loss of mechanical properties at an early time point during degradation that often causes systemic and/or local reactions along with adverse responses (see, Gunatillake, P. A et al., Eur. Cells Mater. 2003, 5, pp. 1-16). Among all polymers widely studied, polyurethanes offer several advantages in designing biodegradable scaffolds. In this context interpenetrating polymer network approach provides several opportunities to tailor the polymeric architecture for tuning dimensional, thermal and mechanical stability along with matrix homogeneity and low degree of crystallinity (see, Park, J. H et al., Molecules 2005, 10, pp. 146-161.; Atala, A et al., Principles of Regen. Med. 2011, p. 1436). However, the mechanisms of cell penetration into these polyurethane scaffolds are lacking in literature.
Biomaterials synthesized using polyurethanes can be made biocompatible, biodegradable with wide range of chemical linkages that help in targeted delivery to specific tissue microenvironment. Synthetically, hydroxyl terminated polymers as starting material for polyurethanes like polycaprolactone, polylactides, polygylcolides, polyethyleneglycols, polyalkyleneadipate, etc. have shown promise as scaffold materials with the polyol based soft segments being hydrolytically degradable (See, Lee, S. I. et al., Biomaterials 2004, 25, pp. 85-96). While biodegradation in polyurethanes can be easily achieved by incorporating hydrolysable moieties like esters in the polymeric chain, control on occasional cytotoxicity of cleaved fragments post-degradation remains the key area of concern and challenge.
To retain structural integrity, porous polyurethane scaffolds are synthesized in network form with intermittent cross-links aiding a 3-dimensional architecture. Often small molecules with three or more functional groups (f>3) conventionally used as cross-linkers can lead to increased toxicity upon degradation. One way to troubleshoot is using larger molecules with active functional groups, which however in most cases can introduce a lot of hard segments difficult to be fragmented under mild conditions. In this context, vegetable oil based polyurethanes, in particular, using castor oil in the polymeric architecture holds promise for future. Castor oil is a vegetable triglyceride with major constituent as ricinoleic acid, a trihydroxyl containing fatty acid (See, Baber, T. M. et al., J Chem. Eng. Data 2002, 47, pp. 1502-1505). Bio-availability from renewable agricultural resource, low cost, low toxicity, traditional medicinal use as laxative and antioxidant makes it an attractive starting material for biomedical polyurethanes under discussion. Long chains can potentially add to the flexibility in a network, ester groups as labile hydrolysable groups, inherent double bonds in combination provide for anti-oxidative properties while the free trihydroxyl functionality can be used as such for urethanation. Hydrophobicity of castor oil was effectively counter balanced by incorporation of exceedingly hydrophilic polyethyleneglycols as chain extenders in the framework. Although, the concept of using vegetable oils has increasingly gained footing in renewable polymer research (See, Biermann, U et al., Angew. Chem. Int. Ed. 2000, 39, pp. 2206-2226). Surprisingly, relatively very few studies have been reported on its biodegradability under physiological conditions and almost none on the use of such polyurethanes as tissue regeneration scaffolds (See, Yeganeh, H. et al., Polym. Degrad. Stab. 2007, 92, pp. 480-489).
To assess the various properties of the polymer network, biocompatibility must be studied in vitro. In vitro models based on immortalized or cancer cell lines provide us the necessary information about cell interactions with polymers. Information such as direct effect of polymer on cell adhesion, proliferation and viability can be investigated during in vitro experiments that are often performed in a single variant controlled environment as compared to in vivo. In-spite of cell lines being immortalized they often needs to overcome issues such as supply of cells, heterogeneity, ease of culture, and fast growth-rate. Studies using more than one cell types in in vitro models are more representative of in vivo native tissue as compared to the use of single cell types (See, Holy, C. E et al., J. Biomed. Mater. Res. 2000, 51, pp. 376-382).
Cells are known to remodel their surrounding extracellular matrix by secreting catabolic enzymes, such as MMPs. A strict balance between these MMPs and TIMP occurs in migrating cells that maintains the proteolytic microenvironment (See, Jennifer, L. W et al., Macromol. 1999, 32, pp. 241-244). Tan et al. Carcinogenesis 2009, 30, pp. 258-268, reported RGD peptide, αvβ3 monoclonal antibody and inhibitors of mitogen-activated protein kinase inhibited the cysteine rich 61-induced increase of cell migration and MMP-13 up-regulation in chondrosarcoma cells suggesting migration of cells by increasing MMP-13 expression through αvβ3 integrin receptor and Erk/MAPK signal transduction pathway. Also reports suggest that Erk/MAPK signalling has direct correlation with the expression of MMP-2 in MDA-MB-231 cells. Further reports suggest an activation of Akt via its phosphorylation mediates MMP-2 expression and thereby promoting cell invasion and proliferation (see, Shuvojit, Moulik J. et al., Tumor 2014, 2, pp. 87-98; Luciana, R. G. et al., BMC Cancer 2012, 12, p. 26; Peter Storz. et al., Mol. Cell. Biol. 2009, 29, pp. 4906-4917).
Oxidative stress plays a crucial role in the pathophysiology of tissue-degenerative diseases. An increase in the production of ROS by injured cells is observed at sights of tissue injury. In events of cell transplantation, transplanted cells are often unable to fight against free radicals at the tissue microenvironment (See, Rahman, T et al., Adv. Biosci. Biotechnol. 2012, 3, pp. 997-1019). Inflammation plays a crucial role in wound healing with evidences of both enzymatic and non enzymatic depletion of antioxidant defenses during the initial 7 days post-wounding (See, Rasik, A. M. et al., Int. J Exp. Pathol. 2000, 81, pp. 257-263). Hypoxic microenvironment at the wound site of cutaneous injury leads to an increased production of ROS and release of certain chemo-attractants by infiltration of activated inflammatory cells like leukocytes, neutrophils, monocytes, leucocytes and lymphocytes as cellular defense mechanism. (See, Steilinga, H. et al., Exp. Cell Res. 1999, 247, 2, pp. 484-494). Following the phase of hemostasis, acute inflammatory phase begins with recruitment of macrophages which secretes pro-inflammatory cytokines. Sustained recruitment of these inflammatory cells leads to chronic wounds (See, Wu, Y. et al., Stem Cells 2010, 28, pp. 905-915). Early phase of inflammation involves the recruitment of neutrophils which gets activated by pro-inflammatory cytokines such as IL-1β, TNF-α, and IFN-γ at the site of injury (see, Rosenberg, L. et al., eMed. Plastic Surg. 2006, 457, pp. 3-6). During the healing process of an injured tissue, re-epithelialization and granular tissue formation which consists of endothelial cells, macrophages and fibroblasts contributes towards tissue integrity. There are evidences of temporal and spatial change in leukocytes subsets during skin wound formation (Sabine, A. E. et al., J. Invest. Dermatol. 2007, 127, pp. 514-525). A decline in the number of inflammatory cells and pro-inflammatory cytokines during progress of healing state indicate an initialization of proliferative phase. Studies in murine wound models where the mice deficient with neutrophils, IFN-γ, TNFRp55 showed accelerated wound healing whereas with depletion in IL6 and TNF-α impaired the healing (Lin, Q. et al., J. Immunol. 2011, 186, pp. 3710-3717). The changing pattern of cytokines released from pro- to anti-inflammatory process initiates the proliferative and remodeling phase of wound healing (See, Newton, P. M. et al., Inflamm. 2004, 28, pp. 207-214).
Hence an ideal polymer host must satisfy the criteria such as (i) stability and biocompatibility; (ii) biodegradability and cellular penetration, and (iii) Must hold anti-oxidant and anti-inflammatory properties thereby cells can be protected against ROS and inflammatory cytokines at the site of the injury.